Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1975.tb02238.x

Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography.

5-Noncoding sequences have been tabulated for 211 messenger RNAs from higher eukaryotic cells. The 5'-proximal AUG triplet serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine (most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine in that position. There is a predominance of C in positions -1, -2, -4 and -5, just upstream from the initiator codon. The sequence CCAGCCAUG (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in a given mRNA matches the -1 to -5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides in common with the CCACC consensus, but only ten mRNAs conform perfectly.

Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs

A major developmental transition in early xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage

Cyclin: A protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division

We describe a method for the synthesis of microgram quantities of eucaryotic messenger RNAs. Injection into the cytoplasm of frog oocytes and addition to wheat germ extracts show that these synthetic RNAs function efficiently as messenger RNAs. We confirm that a 5' cap on the mRNA is essential for translation in injected oocytes and show that most of the 3' flanking region, including the poly A tail, can be deleted without the abolition of protein synthesis. The method of mRNA synthesis involves in vitro transcription of cDNAs which have been cloned into SP6 vectors (described in the accompanying paper). This method enables one to produce large amounts of mRNA and consequently protein from any cDNA clone.

Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs

Injected frog eggs and oocytes provide a very sensitive assay system for the identification of messenger RNA and permit the study of translational control in living cells. The translation of each haemoglobin messenger RNA molecule once every 5–10 minutes for at least 24 hours makes it possible to recognize less than 1 ng of this messenger RNA.

Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells

Xsox17α and -β Mediate Endoderm Formation in Xenopus

The synthesis and storage of histones during the oogenesis of Xenopus laevis

mRNA encoding the T-box transcription factor VegT is located throughout the vegetal pole of the Xenopus egg and is believed to play an important part in endoderm and mesoderm formation. We find that VegT generates endoderm both by cell-autonomous action and by generating TGF-beta family signals, the latter being entirely responsible for its mesoderm-inducing activity. Signalling molecules induced cell-autonomously by VegT include derriere, Xnr4 and activin B. Xnr1 and Xnr2 are also induced, but primarily in a non-autonomous manner. All of these signalling molecules are found in the blastula and gastrula vegetal pole and induce both endoderm and mesoderm in the animal cap assay, and hence are good candidates both for the endogenous zygotic mesoderm-inducing signal and for reinforcing the vegetal expression of endoderm markers.

Hugh R. Woodland

Papers

Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells

Xsox17α and -β Mediate Endoderm Formation in Xenopus

The synthesis and storage of histones during the oogenesis of Xenopus laevis

Mode of action of VegT in mesoderm and endoderm formation.

Xpat, a gene expressed specifically in germ plasm and primordial germ cells of Xenopus laevis